SHIP 1 modulators

ABSTRACT

The present invention includes the use of pelorol, related compounds and pharmaceutical compositions thereof as modulators of SHIP 1 activity. This invention also provides novel terpene compounds capable of modulating SHIP 1 activity and methods of synthesis thereof.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to SHIP 1, a negative regulator ofcell proliferation and survival and immune cell activation.

BACKGROUND OF THE INVENTION

[0002] SH₂-containing inositol 5-phosphatase (SHIP 1), selectivelyhydrolyzes the 5-phosphate from inositol 1,3,4,5-tetraphosphate (IP4)and phosphatalidylinositol 3,4,5-triphosphate (PIP3). U.S. Pat. No.6,238,903 discloses that SHIP 1 is an enzyme regulator of signalingpathways that control gene expression, cell proliferation,differentiation, activation, and metabolism, particularly of the Ras andphospholipid signaling pathways. SHIP 1 plays an important role incytokine and immune receptor signal tansduction. SHIP 1 disrupted (SHIP1−/−) mice exhibit a myeloproliferative phenotype characterized byoverproduction of granulocytes and macrophages¹. SHIP 1−/− mast cellsare more prone to IgE and Steel factor induced degranulation, while SHIP1−/− B cells are resistant to negative regulation by Fc RIIB. SHIP 1 isalso involved in the pathogenesis of chronic myelogenous leukemia².

[0003] Compounds that specifically modulate the activity of SHIP 1 wouldbe useful in the treatment of cell proliferation, hematopoietic andimmune disorders, as well as for manipulating SHIP 1 mediated pathwaysduring investigatory and drug discovery testing. To date, no structureof a small molecule SHIP 1 specific modulator has been disclosed.

[0004] A sesquiterpene compound termed pelorol may be obtained fromvarious marine sponge species, including Petrosaspongia metachromia andDactylospongia elegans. Kwak et al. and Goclik et al. each disclosed thestructure of pelorol and its extraction from different marinesponges.^(4,5) Pelorol was reported as having weak antitrypanosomal andantiplasmodial effects⁵. The precise structure of pelorol is as follows,with Me representing a methyl group and relative configuration of chiralatoms (C-5, 8, 9 and 10) shown.

[0005] Some reduced and substituted chrysene derivatives similar topelorol and having the characteristic gem substituted non-aromatic ringof pelorol are known as intermediates or derivatives in the preparationof various polycyclic polyprenols found in shale⁶⁻¹², in the preparationof taxodione¹³, and in the compound1,2,3,4,4a,4b,5,6,10b,11,12,12a-dodecahydro-1,1-dimethyl-chrysene¹⁴.None of these chrysene derivatives are known to have biologicalactivity.

SUMMARY OF THE INVENTION

[0006] This invention is based on the discovery that pelorol and relatedcompounds are capable of modulation of SHIP 1 activity.

[0007] Some embodiments of this invention provide novel compounds ofFormula I and salts thereof. Compounds of Formula I have the structure:

[0008] wherein;

[0009] R₁ and R₂ are independently selected from the group consistingof: —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′;

[0010] R₃ and R₄ are independently selected from the group consistingof: H, —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′;

[0011] Q is selected from the group consisting of: —CH₂—, —CY₁Y₂—,—CH₂CH₂—, —CH═CH—, —CY₁Y₂CY₃Y₄—, —CH₂CH₂CH₂—, —CH═CHCH₂—, —CH═CHCY₁Y₂—,and —CY₁Y₂CY₃Y₄CY₅Y₆—; where Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆ areindependently selected from the group consisting of: H, F, Br, Cl, I,OH, OR′, and SH; or any one group of Y₁/Y₂, Y₃/Y₄, and Y₅/Y₆may be ═O;or Y₁/Y₃ may form an epoxide; and, at least one of Y₁, Y₂, Y₃, Y₄, Y₅and Y₆ when present, is not H;

[0012] X₁, X₂, X₃, and X₄ are independently selected from the groupconsisting of: H, R, OH, —OR, —CO₂H, —CO₂R′, F, Br, Cl, I, —CN, —SO₃H,—OSO₃H, NO₂, NH₂, —NHR, and —NR₂; where R is a linear, branched, orcyclic, saturated or unsaturated one to ten carbon alkyl group that isunsubstituted or is substituted with one or more of: OH, ═O, SH, F, Br,Cl, I, NH₂, —NHR′, —NR′₂, NO₂, —CO₂H, —CO₂R′, and epoxide;

[0013] and R′ is a linear, branched, or cyclic, saturated or unsaturatedone to ten carbon alkyl group that is unsubstituted or substituted withone or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR″, —NR″₂, NO₂ and—CO₂H where R″ is a linear, branched, or cyclic, saturated orunsaturated one to ten carbon alkyl group.

[0014] Novel compounds of Formula I of this invention do not include theprecise structures of previously described gem substituted chrysenederivatives. These previously described compounds include pelorol andcompounds having the following structures in which Me is methyl:

[0015] Alternately defined, this invention excludes such previouslyknown specific compounds of Formula I in which each of R₁-R₄ are methyl;Q is —CH₂CH₂—; and, X₁-X₄ is according to any one of the followingdefinitions:

[0016] (a) X₁ and X₂=OH, X₃=H, and X₄=—COOCH₃;

[0017] (b) X₁, X₂, X₃ and X₄=H;

[0018] (c) X₁, X₂, and X₄ H, and X₃=CH₃;

[0019] (d) X₁, X₃, and X₄=H, and X₂=CH₃;

[0020] (e) X₂, X₃, and X₄=H, and X₁=CH₃; and

[0021] (f) X₁ and X₄=H, X₂ and X₃=OCH₃.

[0022] Also excluded is a compound of Formula I in which R₁ and R₂=CH₃;R₃ and R₄=H; Q=—CH₂CH₂—; and each of X₁-X₄ is H.

[0023] Some embodiments of this invention provide a pharmaceuticalcomposition comprising one or more compounds of Formula I orpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier. Such compositions may comprise previously knowncompounds of Formula I which have not been known as biologically activecompounds suitable for pharmaceutical use.

[0024] Some embodiments of this invention provide a method of treatmentor prevention of an immune, inflammatory, or neoplastic disorder orcondition, comprising administering to a patient in need of suchtreatment or prevention, an effective amount of a compound of Formula Ior pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition of this invention.

[0025] Some embodiments of this invention provide the use of a compoundof Formula I or pharmaceutically acceptable salt thereof for modulationof SHIP 1 activity and for preparation of agents for the modulation ofSHIP 1 activity. Such modulation may be in vitro or in vivo. Agents forin vivo use include a pharmaceutical composition of this invention aswell as agents adapted for in vitro use. The modulation may be for atreatment or prevention of an immune, inflammatory, or neoplasticcondition or disorders as described above.

[0026] Some compounds of Formula I may be prepared in whole or in partby fractionating biological extracts or by derivatizing availablecompounds. Alternately, compounds of Formula I may be prepared by totalsynthesis.

[0027] Some embodiments of this invention provide a method of making acompound of Formula IA

[0028] in which R₁-R₄, X₁, X₃, and X₄ are as defined for Formula I, L′is a C₁-C₄ saturated or unsaturated alkyl linking group; and A is anactivating group; comprising reacting a compound of Formula IIA or IIB:

[0029] in which L is absent or is a C₁-C₃ saturated or unsaturated alkyllinking group and E and E′ are electrophilic reactive groups; with acompound of Formula III

[0030] in which Nu is a group that renders the compound of Formula IIInucleophilic at Nu, followed by optional reduction and by hydrolysis, toproduce a compound of Formula IV

[0031] and condensing the compound of Formula IV to produce a compoundof Formula IA.

[0032] L′ in compounds of Formula IA may optionally be changed orderivatized to form a desired component Q of Formula I. For example,component L′ in compounds of Formula IA produced by the preceding methodmay have different degrees of saturation or different substituents ascompared to Q in a compound of Formula I. In order to reduce the numberof atoms in the ring, a compound having an unsaturated L′ group could besubjected to oxidizing and reduction steps to reduce the size of thering in Formula I comprising Q. In addition, functionalities such asketone, hydroxyl, or other groups may be added to L′ to form a desired Qcomponent.

[0033] Preferred electrophilic reactive groups for E are lactone, ester,and thioester. A preferred group for E′ is carboxyl. More preferably,compounds of Formulas IIA and IIB are as follows.

[0034] Even more preferably, compounds of Formulas IIA and IIB are asfollows:

[0035] A preferred Nu in compounds of Formula III is lithium which maybe substituted onto the ring for a halogen such as bromine. Preferably,A in the compound of Formula III is an activating group such as —OMe orNHAc (Me=methyl and Ac=acetyl) which group may be subsequently convertedto a desired substituent for X₂ in compounds of Formula I. Substitutentsmay also be protected, where appropriate with a protecting group such asTBS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 is a graph depicting the effect of sponge extracts on SHIP1 enzyme activity in vitro.

[0037]FIG. 2 is a graph depicting the effect of pelorol on macrophagenitric oxide (NO) production.

[0038]FIG. 3 is a graph depicting the effect of pelorol on IgE mediatedmast cell activation.

DETAILED DESCRIPTION OF THE INVENTION

[0039] In this specification, the following abbreviations will appear:THF (tetrahydrofuran); n-buLi (n-butyly lithium); t-buLi (tert-butylylithium); Ph₃PMe (methyl triphenyl posphonium bromide); PCC (pyridiniumchlorochromate); Ac (acetyl); Me (methyl); Et (ethyl); prop. (propyl);but. (butyl); RT or, r.t. (room temperature); hr. (hour(s)); DMSO(dimethylsulfoxide); DNFB (2,4-dinitrofluorobenzene); LPS(lipopolysaccarhide); TNF-α (Tumor Necrosis Factor Alpha); TBS(tert-butyl dimethylsilyl); and EA (ethyl acetate).

[0040] SHIP 1 Modulating Compounds

[0041] Compounds of Formula I have chiral centres at C-5, C-8, C-9 andC-10 and may be chiral at C-4 depending upon whether R₁ and R₂ aredifferent. Compounds of this invention include all stereoisomers andenantiomers of compounds of Formula I. Some embodiments have the samerelative configuration of chiral centres as does pelorol or areenantiomers thereof, namely: S, R, R, S; or R, S, S, R (at C-5, 8, 9 and10 respectively). Some embodiments have the same absolute configurationas pelorol at chiral centres. Some embodiments have the same relativeconfiguration as pelorol at C-5 and C-10 with independently variableconfigurations at C-8 and C-9. Some embodiments have the same relativeconfiguration as pelorol at C-5, C-8, and C-10 with variableconfiguration at C-9. In all cases, the configuration at C-4 (if chiral)may be variable or may be the same relative configuration to theremaining chiral centres as is shown in examples of structures ofcompounds of Formula I illustrated herein.

[0042] In various embodiments of this invention, the compounds may havethe limitations in Formula I described above or may have more specificlimitations with respect to substituents Q, R₁-R₄, and X₁-X₄. Anycombination of the following limitations is encompassed by thisinvention.

[0043] (a) Q may be as defined for Formula I except that Y₁₋₆ is limitedto H or halogen;

[0044] (b) Q may be limited to —CH₂—, —CH₂CH₂—, —CH═CH, —CH₂—CH₂CH₂— and—CH═CHCH₂—;

[0045] (c) Q may be limited to H or saturated moieties in the limitationof Formula I, or according to the limitations of paragraph (a) or (b)above;

[0046] (d) Q may be limited to a one or two carbon skeleton within thelimitations of Formula I, or according to the limitations of any ofparagraphs (a)-(c) above;

[0047] (e) one or both of R₁ and R₂ may be limited to methyl, ethyl,—CH₂OH or —CH₂OR′;

[0048] (f) R′ in one or both of R₁ and R₂ according to Formula I, or thelimitation of paragraph (e) above, may be limited to methyl, ethyl,propyl or butyl;

[0049] (g) one or both of R₁ and R₂ may be limited to methyl or ethyl;

[0050] (h) one or both of R₁ and R₂ may be limited to methyl;

[0051] (i) R and R′ in any one or more of X₁-X₄ may be limited tounsubstituted methyl, ethyl, propyl or butyl;

[0052] (j) one or more of X₁-X₃ may be limited to H, R, OH, OR, halogen,—CONH₂, —CONHR′, —COR′₂, NHR or NR₂ where R and R′ are limited as inFormula I, or R and R′ may be according to paragraph (i) above;

[0053] (k) one or more of X₁-X₃ is limited to H, OH, OR, —CONH₂,—CONHR′, and —COR′₂, where R and R′ are as in Formula I, or R and R′ maybe limited according to paragraph (i) above;

[0054] (I) one or more of X₁-X₃ may be limited to H, OH, and OCH₃;

[0055] (m) X₄ may be limited to H, R, OH, OR, CO₂H or —CO₂R′, with R andR′ as in Formula I, or R and R′ may be limited according to paragraph(i) above;

[0056] (n) X₄ may be limited to H, R, OH, OCH₃, —CO₂H and —CO₂R′ with Rand R′ limited according to paragraph (i) above; and,

[0057] (o) X₄ may be limited to H, R, OH, OCH₃, —CO₂H or —CO₂CH₃.

[0058] The following specific structures are embodiments of thisinvention. In some cases, variability at X₁, X₂, and X₄ is shown withreference to substituents identified as R, Z, and Y, which for thepurposes of the illustrated compounds are defined below. Althoughrelative stereochemistry is illustrated for each structure, theconfiguration of chiral centres may vary according to any of theembodiments based on chirality described above.

[0059] Sources of Compounds and Assays for Activity

[0060] Pelorol may be obtained from natural sources as taught in theprior art and in the Example 1 herein. Solvent fractionation and/orchromatography may be employed. It is also possible to modify pelorol orother available compounds such as chrysene derivatives by known chemicalmethodologies to add, remove, or replace substituents in order toproduce components of Formula I. Examples of such derivatization stepsas applied to different compounds of Formula I are shown in more detailbelow.

[0061] The presence of SHIP 1 modulating compounds in a preparation maybe determined by use of a variety of assays, including direct monitoringof a change in activity of SHIP 1 enzyme such as by the methodologydisclosed in Example 1 and FIG. 1 or by biological assays which may bereadily adapted from known procedures, including cell or animal basedassays which monitor changes in: nitric oxide production from activatedmacrophages; IgE induced mast cell degranulation; LPS induced macrophageactivation; TNF-α expression or activity. In addition, standard assaysfor agents which mediate inflammatory activity in living subjects may beemployed. Adaptation of these assays is facilitated by the availabilityof SHIP 1^(−/−) and SHIP 1^(+/−) mice^(15,16) and bone marrow derivedmacrophages¹⁷. In addition, the availability of anti-SHIP 1 antibodies¹⁸facilitates use of immunoassay formats. Such assays may also be used toassess activity of compounds prepared by total synthesis, as describedherein.

[0062] Total Synthesis of Compounds

[0063] A synthetic scheme for making pelorol and other compounds ofFormula I is provided herein. Tables (1-2) provide detailed examples oftwo embodiments of such a synthesis with examples of different compoundsof Formula I which may be prepared. The compound shown in the Tablesthat is identical to pelorol except that the ring adjacent the aromaticring has six members, is termed “homopelorol”. Compounds having asix-membered ring are termed “homopelorol analogs”. Compounds having afive-membered ring other than pelorol are termed herein, “pelorolanalogs”.

[0064] In the synthesis methods shown in Tables 1 and 2, compounds ofFormula IIA shown therein are conveniently based on sclareolide as astarting material. Appropriate derivatives of sclareolide providingdesired R₁-R₄ substituents may be employed. In the aromatic compound ofFormula III shown in the Tables, Nu is preferably lithium. X₂ in thestarting compound of Formula III is preferably an activating group suchas —OMe or —NHAc. X₁-X₄ may remain as found in the starting material orbe appropriately altered to provide the desired substituents for the endproduct. Protecting groups may be employed on R₁-R₄ or X₁, X₃, or X₄. Anexample of derivatization of the ring comprising L′ in Formula IA toproduce a desired component Q of Formula I is illustrated in Table 2where oxidation (e.g. by treatment with OsO₄ followed by treatment withan acid such as HCl) is performed to provide a ketone substituent on thering. TABLE 1 Synthesis of Pelorol and Pelorol Analogs

[0065] TABLE 2 Synthesis of Homopelorol and Homopelorol Analogs

Pharmaceutical Compositions, Dosages, Administration and Indications

[0066] Compounds for use in this invention may be formulated intopharmaceutical compositions in any number of ways, which would be knownto a person of skill in the art, all of which are within the scope ofthe invention. The person of skill in the art may be expected to selectappropriate pharmaceutically acceptable salts as well as appropriatepharmaceutically acceptable excipients, diluents, and carriers.

[0067] Compounds according to the invention can be provided alone or incombination with other agents (for example, small molecules, peptides,or peptide analogues) in therapeutically- or prophylactically-acceptableamounts, in any pharmaceutically acceptable carrier. Methods well knownin the art for making such pharmaceutical formulations are found in, forexample, “Remington's Pharmaceutical Sciences” (19^(th) edition), ed. A.Gennaro, 1995, Mack Publishing Company, Easton, Pa., incorporated byreference herein. Pharmaceutical formulations according to the presentinvention may, for example, contain excipients, sterile water, orsaline, ethanol, methanol, dimethyl sulfoxide, polyalkylene glycols suchas polyethylene glycol, propylene glycol, or other synthetic solvents,oils of vegetable origin, or hydrogenated napthalenes.

[0068] Compounds according to the invention may include hydrophobiccompounds, for example, compounds that are substantially insoluble inwater, but are freely soluble in solvents such as, for example, ethanol,methanol, dimethyl sulfoxide, or chloroform, or combinations thereof.Formulations containing such hydrophobic compounds may be providedusing, for example, micelles, which are formed by amphiphilic compoundsunder certain conditions. In aqueous solutions, micelles are capable ofincorporating hydrophobic compounds in their hydrocarbon cores, orwithin the micelle walls. Hydrophobic compounds may also be provided bysolubilization in triglycerides (oils), for example, a digestiblevegetable oil. The solubilized hydrophobic compound in the oil phase maybe dispersed in an aqueous solution and stabilized using emulsifyingagents, if desired. Alternatively, the hydrophobic compound may beprovided in oil and delivered, for example, to the gastrointestinalsystem where bile salts may function as in vivo emulsifiers. Hydrophobiccompounds may also be provided as microemulsions which, like emulsions,are liquid dispersions of oil and water, but have smaller particles withan oil phase in a micelle-like “core.” Hydrophobic compounds accordingto the invention may also be provided together with a polymeric carrier,for example, a carbohydrate such as starch, cellulose, dextran,cyclodextrin, methylcellulose, or hyaluronic acid, or a polypeptide,such as albumin, collagen, or gelatin. Other modes of formulation ofhydrophobic compounds may include liposomes, natural and syntheticphospholipids, or solvents, for example, dimethyl sulfoxide or alcohols.

[0069] The pharmaceutical compositions of the invention may beformulated so as to provide controlled release of the active compound(s)over a period of time. Thus, the formulations could contain, forexample, an amount of the compound that would be toxic if administeredas a single dose, but whose controlled release does not exceed toxiclevels. Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers, for example,may be used to control the release of the compounds. Other potentiallyuseful delivery systems for modulatory compounds according to thepresent invention include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes.

[0070] A “therapeutically effective amount” of a compound is an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result using a compound according to the invention.A therapeutically effective amount is also one in which any toxic ordetrimental effects of the comopund are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” of a compound refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic result.Typically, a prophylactic dose is used in subjects prior to or at anearlier stage of disease, so that a prophylactically effective amountmay be less than a therapeutically effective amount. Amounts consideredsufficient will vary according to the specific compound used, the modeof administration, the stage and severity of the disease, the age, sex,weight, and health of the individual being treated, and concurrenttreatments.

[0071] A preferred range for therapeutically or prophylacticallyeffective amounts of the compounds of the invention may be 0.1 nM-0.1M,0.1 nM-0.05M, 0.05 nM-15 μM or 0.01 nM-10 μM. It is to be noted thatdosage values may vary with the severity of the condition to bealleviated. For any particular subject, specific dosage regimens may beadjusted over time according to the individual need and the professionaljudgement of the person administering or supervising the administrationof the compositions. Dosage ranges set forth herein are exemplary onlyand do not limit the dosage ranges that may be selected by medicalpractitioners. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation.

[0072] In general, compounds of the invention should be used withoutcausing substantial toxicity. Toxicity of the compounds of the inventioncan be determined using standard techniques, for example, by testing incell cultures or experimental animals and determining the therapeuticindex, i.e., the ratio between the LD50 (the dose lethal to 50% of thepopulation) and the LD100 (the dose lethal to 100% of the population).In some circumstances however, such as in severe disease conditions, itmay be necessary to administer substantial excesses of the compositions.

[0073] Conventional pharmaceutical practice may be employed to providesuitable formulations or compositions to administer the compounds topatients, depending on the therapeutic or prophylactic objectives. Anyappropriate route of administration may be employed, for example,systemic, parenteral, intravenous, subcutaneous, transdermal,transmucosal, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracistemal,intraperitoneal, intranasal, aerosol, topical, surgical, or oraladministration. The formulations used may vary according to the chosenroute of administration. Thus, for oral administration, the formulationsmay be in the form of tablets or capsules; for inhalants, theformulations may be in the form of powders, nasal drops, or aerosols;for transmucosal administration, the formulations may be nasal sprays orsuppositories; for transdermal administration, the formulations may becreams, ointments, salves, or gels; etc.

[0074] Therapeutically effective or prophylactically effective amountsof SHIP 1 modulators and pharmaceutical compositions of this inventionmay be administered to patients in need of treatment or prophylaxis forcancer (neoplastic diseases), other cell proliferative disorders,inflammatory diseases and immune diseases. Neoplastic diseases includebut are not limited to: leukemias, carcinomas, sarcoma, melanomas,neuroblastoma, capillary leak syndrome and hematological malignancies.Diseases with an inflammatory component include, but are not limited to:rheumatoid arthritis, multiple sclerosis, Guillan-Barre syndrome,Crohn's disease, ulcerative colitis, inflammatory bowel syndrome,psoriasis, graft versus host disease, host versus graft, lupuserythematosis, Alzheimer's disease and insulin-dependent diabetesmellitus. Diseases related to inappropriate activation ofmacrophage-related cells of the reticuloendothelial lineage includeosteoporosis.

[0075] Pelorol and other compounds having the structure of Formula Iexhibit SHIP 1 agonist activity. By activating SHIP 1, such agonists areparticularly useful in the treatment of inflammatory diseases such assepsis/septic shock, colitis, inflanunatory bowel syndrome, and thoseinvolving macrophage proliferation or activation; neoplastic diseasessuch as myeloid and lymphoid leukemias; as an immunosuppressive agentsuch as in transplant rejection; hematopoietic disorders; and foraffecting mast cell degeneration such as in the treatment or preventionof allergies.

EXAMPLE 1

[0076] In a preliminary screen of 150 marine organism extracts, extractswhich activated SHIP 1 in an enzyme assay were identified. Assay-guidedfractionation of one of these extracts resulted in the identification ofthe active compound as being pelorol (FIG. 1). The origin and processingof the extracts which tested positive in the screen and the nature ofthe assay were as follows.

[0077] Specimens of the brownish sheet sponge Dactylospongia elegans(order Dictyoceratida, family Spongiidae) were collected by hand usingSCUBA at a depth of 5-10 m from a protected overhang in Rasch Passage onthe outer reef of Madang Lagoon, Papua New Guinea, in January 1995.Freshly collected sponge was frozen on sight and transported toVancouver, Canada over dry ice. The sponge was identified and forverification, a voucher sample was placed in the Zoological Museum ofAmsterdam (ZMA POR. 15986). The frozen sponge (120 g) was cut into smallpieces, immersed in and subsequently extracted repeatedly with MeOH(3×250 mL). The combined methanolic extracts were concentrated in vacuoand then partitioned between EtOAc (4×100 mL) and H₂O (300 mL). Thecombined EtOAc extract was evaporated to dryness in vacuo to yield 490mg of a brownish purple oil, found to contain pelorol.

[0078] The assay was performed in 96-well microtitre plates. SHIP 1enzyme was produced with a hemagglutinin and a hexahistidine tag, from amammalian expression vector. The His tag was employed to enhancepurification. SHIP 1 enzyme (10 ng) was incubated with extract or DMSOfor 15 minutes at room temperature before addition of 200 Minositol-1,3,4,5-tetrakisphosphate. The reaction was allowed to proceedfor 20 minutes at 37 degrees C. The amount of inorganic phosphatereleased was then assessed by the addition of malachite green reagentfollowed by an absorbance measurement at 650 nm.

EXAMPLE 2

[0079] Pelorol was prepared according to the following scheme, under thespecific conditions described below.

[0080] To a stirred solution of 1 (1.00 g, 3.99 mmol) in anhydrous Et₂O(30 mL) was added a freshly prepared 1.6M solution of MeLi in Et₂O (3mL, 4.8 mmol) in portions for 10 min at r.t. and stirring was continuedfor another 5 min. The mixture was then treated with 10% HCl (2 mL),then transferred to a funnel and extracted with ethereal repeatedly. Thecombined extracts was washed with NaHCO₃ and H₂O, dried (MgSO₄),filtered and concentrated. The residue was column chromatographed withhexane/Et₂O (6:4) to give 0.74 g (70%) of 2.

[0081] To a stirred, cooled (ice bath) solution of (CF₃CO)₂O (9 mL,63.85 mmol) in CH₂Cl₂ (40 mL) was added 50% aq H₂O₂ (1.8 mL, 31.66 mmol)and the mixture was allowed to stand in an ice bath for 10 min. Allsubsequent operations were performed at r.t. The solution was treatedwith solid NaHCO₃ (5.40 g, 64.28 mmol) for 2 min and after stirring themixture for 8 min, a solution of 2 (1.80 g, 6.76 mmol) in CH₂Cl₂ (54 mL)was added. The resulting mixture was stirred for 30 min and then, afteraddition of H₂O (10 mL), was treated with solid NaHCO₃ in portions for45 min until the pH reached 7. Finally, the mixture was extracted withEt₂O. The combined extracts were washed with NaHCO₃, H₂O and dried(MgSO₄), filtered and concentrated to give pure 3.

[0082] Compound 3 (1 g, 3.6 mmol) was dissolved in 10% solution of KOHin MeOH (1 mL, 1.78 mmol) at 0° C. The resulting mixture was stirred for10 min. After addition of H2O, the solution was extrated with Et2O. Theextrated was washed with H₂O, dried (MgSO₄), filtered and concentratedto give 0.8 g of 4.

[0083] In an oven dried , N₂ flushed 100 mL round bottom flask equippedwith a magnetic stirring bar was placed 3.24 g(15 mmol) of PCC, 30 mL ofCH₂Cl₂ and 2.4 g (10 mmol) of 4. The mixture was well stirred at r.t.for 2 hrs and was quenched by adding 30 mL of Et₂O. The resultingsolution was filtered through a thick pad of silica gel and concentratedto give a residue. The residue was column chromatographed with hexane/EA(8:2) to give 1.6 g (67%) of 5.

[0084] Sodium hydride (24.6 mg, 0.82 mmol, 80% oil dispersion) and dryTHF (5 mL) were added to a dry flask equipped with a condenser and dryN₂ flow. To this suspension was added methyl triphenyl phosphoniumbromide (0.146 g, 0.41 mmol) and the mixture was stirred for 10 min.Then 6 (100 mg, 0.41 mmol) in THF (2 mL) was added and the mixture wasgently reflux for 2 h. The reaction was quenched by adding 2 mL ofmethanol and then extracted with Et₂O. After usual work up treatment.94.3 mg of 7 was afforded.

[0085] A 1.6M solution of tBuLi in pentane (1.74 mL, 2.79 mmol) wasadded slowly to a stirred solution of 7 (612.6 mg, 2.52 mmol) in dry THF(20 mL) at −78° C. After stirring for 30 min, a solution of 5 (300 mg,1.26 mmol) in dry THF (5 mL) was added. The mixture was further stirredat −78° C. for 2 hrs. Then H₂O (10 mL) was added and the mixture wasextracted with Et₂O (120 mL twice). The combined Et₂O extracts werewashed with sat.brine, dried (MgSO₄) and concentrated to give a residue,which was chromatographed on NP Sepak™ to give 280 mg (55%) of 9.

[0086] A solution of 9 (40 mg, 0.1 mmol) in EA (5 mL) was hydrogenatedover 10% Pd/C (50 mg) under an atmosphere of hydrogen at r.t. overnight. Filtration and concentration gave 37 mg (96%) of 10.

[0087] To a stirred solution of 10 (38.8 mg, 0.1 mmol) in CH₂Cl₂ (10mL), SnCl₄ (0.1 mL) was added slowly at −20° C. under argon for 2 min.The resulting mixture was further stirred for 20 min and then dilutedwith CH₂Cl₂ (20 mL) and poured into ice. The aqueous phase was extractedwith CH₂Cl₂ twice (20 mL) and combined the extracts, washed withsaturated NaHCO3, saturated brine and dried over MgSO₄. Evaporation toafford 11 (28 mg, 76%).

[0088] PCC (41.6 mg, 0.192 mmol) was added to 11 (7.4 mg, 0.02 mmol)dissolved in 2 mL of CH₂Cl₂. The mixture was stirred at gentle refluxfor 24 hrs under Argon. The reaction was diluted with Et₂O (20 mL) andthe resulting dark solution was filter through a NP Sepak™.Concentration of the filtrates and further purification afford 1.5 mg(20%) of 12.

[0089] 1.5 mg of 12 was dissolved and stirred in 2 mL of NaOH(10%)solution (containing 0.5 mL THF). 5 mg Iodine is added subsequently andthe mixture was further stirred for 20 min and acidified by adding 3 mLof 10% H₂SO₄. The solution was extracted with 50 mL of Et₂O, washed withsaturated brine and concentrated to afford a residue 13.

[0090] 38.6 mg (0.1 mmol) of 13 was stirred in CH₂Cl₂ (1 mL) underArgon. BBr₃ in CH₂Cl₂ (2.0 mL 1M) was added, and stirring was continuedfor 1.5 h. The mixture was then poured into H₂O and extracted withCH₂Cl₂ (50 mL). The combined extracts were then dried over MgSO₄,filtered and concentrated. The residue was purified by NP Sepak™(hexane:EA=7:3) to afford 14 (25 mg, 70%).

[0091] 35.8 mg (0.1 mmol) of 14 was dissolved in MeOH (2 mL) containing5% H₂SO₄. Stirring was continued for 2 hr and the mixture was extractedwith Et₂O, dried over MgSO₄ and concentrated to afford 15.

EXAMPLE 3

[0092] The pelorol analog PNSR-15A was synthesized following themethodologies described in Example 3 using the following scheme.

EXAMPLE 4

[0093] The homopelorol analog PNSR-4A was synthesized by themethodologies described above and according to the following scheme.

EXAMPLE 5

[0094] The homopelorol analog PNSR-14A was synthesized by themethodologies described above and according to the following scheme.

EXAMPLE 6

[0095] Homopelorol may be synthesized according to the following schemebased on the preceding examples and following the methodologiesdescribed above.

EXAMPLE 7

[0096] In addition to causing an increase in activity in the SHIP 1enzyme assay described for FIG. 1, agonist compounds of Formula Iexhibit anti-inflammatory actions on macrophages and mast cells inintact cell-based assays, inhibit nitric oxide production from endotoxinactivated wild-type macrophages and exhibit anti-inflammatory actions onlive subjects. Results obtained for pelorol in NO release and mast cellactivation assays are shown in FIGS. 2 and 3, respectively. Inhibitionof NO release was not observed in SHIP 1−/− macrophages. Pelorolsignificantly reduced IgE induced mast cell degranulation.

[0097] Procedures used in the cell and animal based assays are describedbelow. Results for pelorol and various analogs within Formula I areshown in Table 3, including results using the enzyme assay described inExample 1.

[0098] For the NO release assay, wild-type or SHIP 1−/− macrophage cellswere aliquoted into microtitre plates (5×10⁴/well) and activated with 1g/mL endotoxin (LPS) in the presence or absence of test compound or DMSOcarrier. The cells were incubated at 37° C., 5% CO₂ for 24 hours and theculture supernatant was removed for NO determination using the Griessreagent. Alternatively, J774.1a macrophage cells were treated with 10μg/ml of test compound dissolved in DMSO for 40 minutes prior to theaddition of LPS. Culture supernatants were collected after 24 hr. fordetermination of NO concentration using the Griess reagent.

[0099] For the mast cell activation assay, bone marrow derived mastcells were incubated at 4° C. with anti-DNP IgE for 1 hr. They were thenwashed twice with 23° C. Tyrode's buffer, and incubated in the presenceof test compound or vehicle control for 30 minutes before a 15 minutetreatment with DNP-human serum albumin. The degree of degranulation wasdetermined by measuring the release of β-hexosaminidase.

[0100] For the macrophage TNF-α production assay, J774.1a macrophagecells were treated with 10 μg/mL of test compound dissolved incyclodextrin for 40 minutes prior to the addition of 100 ng/mL LPS.Culture supernatants were collected after 2 hr and 5 hr for TNF-αdetermination by ELISA.

[0101] The mouse ear edema (Evans Blue) assay is a standard model forallergic inflammation. Mice were passively sensitized by intravenousinjection of monoclonal anti-DNP IgE antibody. 24 hours later, 10 μgtest compound (right ears) in 20 μl DMSO:Methanol (1:3) or vehicle alone(left ears) were applied 20 minutes followed by application of theinducing agent [20 μl of 0.15% DNFB in acetone:olive oil (4:1)]. Micewere then injected intravenously with 300 μl 1% Evans Blue. Vascularpermeability was measured at 1 hr after application of the inducingagent by visual inspection and quantification of Evans Blueextravasation in the ear. To quantify the Evans Blue content, ears wereharvested at 1 hr post DNFB treatment and Evans Blue was extracted byincubation in formamide at 37° C. for 24 hr and quantified byspectophotometry at 620 nm. Ears pretreated with carrier alone mounted aprompt anaphylactic reaction in response to DNFB challenge. In contrast,SHIP 1 agonists showed a clear inhibition of vascular permeabilizationas shown by decreased Evans Blue extravasation.

[0102] The mouse ear edema (lymphocyte infiltration assay) is a contacthypersensitivity or ear inflammation model and is a standard in vivomodel for human allergy. Contact hypersensitivity consists of an initialsensitizing phase and an elicitation phase. The latter phase occurs whenthe epidermal cells encounter a particular antigen to which they havepreviously been exposed and is characterized by localized immune cellinfiltration, inflammation, and edema. In this assay, female 4 week old(20 g) Balb/c mice were sensitized to the haptenizing agent,2,4-dinitrofluorobenzene (DNFB) by shaving their abdominal region withan electric razor before applying 25 μl of 0.5% DNFB in acetone:oliveoil (4:1, v/v) to the abdominal wall for two consecutive days. Four daysafter the second application, mice were lightly anesthesized withhalothane before being challenged (treated) epicutaneously on each sideof the right and left ear with 10 μl of 0.2% DNFB. All mice received a500 μl intraperitoneal (i.p.) injection of [³H]-methyl thymidine insterile saline (1 μCi/g body weight) 24 hours before epicutaneouschallenge with DNFB. Thirty minutes prior to DNFB challenge, the rightand left ears were pretreated with test compound in DMSO:methanol (1:3,v/v) or vehicle alone, respectively. Twelve hours following DNFBchallenge, mice were sacrificed by CO₂ asphyxiation and 8 mm diameterplugs were taken from each ear and digested in 500 μl Solvable™ at 60°C. for 10-12 hours. Samples were decolourized by the addition of H₂O₂and analyzed for radiolabelled leukocyte infiltrates by standard liquidscintillation counting.

[0103] The colitis assay is based on determining whether a test compoundprotects mice from TNBS (trinitrobenzene sulfonic acid) inducedinflammation. Test compound (10 mg/kg) or vehicle control was injectedintraperitoneally into mice just prior to a TNBS enema administration.After 2 days, the colons of the vehicle treated mouse were severelyinflamed while the SHIP 1 agonist treated mouse had no signs ofinflammation. TABLE 3 Mouse ear Mouse ear edema SHIP MacrophageMacrophage edema (leukocyte enzyme NO TNFα Mast cell (evans blueinfiltration assay production production activa-tion assay) assay)colitis Pelorol + + + + + + + + + + + + + + + ND + + + Dimeth- + + + +ND ND ND ND ND oxypelorol PNSR-4A insol ND ND ND + + + ND ND PNSR-15A NDND ND ND ND + + ND PNSR-16A ND ND + + + ND ND + + ND PNSR-17A ND ND + +ND ND ND ND PNSR-18A ND ND + + + ND ND ND ND

[0104] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of skill in the artin light of the teachings of this invention that changes andmodification may be made thereto without departing from the spirit orscope of the appended claims. All patents, patent applications andpublications referred to herein are hereby incorporated by reference.

References

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[0107] 3. Lui, Ling et al. (2001) Blood 98:1225 (Abstract No. 1225).

[0108] 4. Kwak, J. H. et al. (2000) J. Nat. Prod. 63:1153-56.

[0109] 5. Goclik, E. et al. (2000) J. Nat. Prod. 63:1150-52.

[0110] 6. Ishihara, Kazuaki et al.(2001) J. of the American Chem. Soc.123:1505-1506.

[0111] 7. Ishihara, Kazuaki et. al. (2002) J. of the American Chem. Soc.124:3647-3655.

[0112] 8. Rosales, Viale et al. (2002) J. of Organic Chem. 67:1167-1170.

[0113] 9. Corey, Elias J. et al. (1998) Angewante Chemie, Intl. Ed.37:1126-1128.

[0114] 10. Boreham, Christopher J. et al. (1995) Organic Geochemistry23:461-6.

[0115] 11. Freeman, Katherine H. et al. (1994) Organic Geochemistry21:1037-1049.

[0116] 12. Schaeffer, Phillipe et al. (1994) Angewante Chemie106:1235-8.

[0117] 13. Harrington, Scott R. et al. (1994) Tetrahedron 50:9229-54.

[0118] 14. Registry Number 112299-69-1

[0119] 15. Helgason, C. D. et al. (1998) Genes Dev. 12:1610.

[0120] 16. Helgason, C. D. et al. (2000) J. Exp. Med. 191:781.

[0121] 17. O'Farrell, A. M. et al. (2000) J. Immunol. 164:4607.

[0122] 18. Damen, J. E. et al. (1998) Blood 92:1199.

1. A compound of Formula I or a salt thereof,

wherein; R₁ and R₂ are independently selected from the group consistingof: —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; R₃ and R₄are independently selected from the group consisting of: H, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; Q is a carbonskeleton selected from the group consisting of: —CH₂—, —CY₁Y₂—,—CH₂CH₂—, —CH═CH—, —CY₁Y₂CY₃Y₄—, —CH₂CH₂CH₂—, —CH═CHCH₂—, —CH═CHCY₁Y₂—,and —CY₁Y₂CY₃Y₄CY₅Y₆—; where Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆ areindependently selected from the group consisting of: H, F, Br, Cl, I,OH, OR′, and SH; or any one group of Y₁/Y₂, Y₃/Y₄, and Y₅/Y₆ may be ═O;or Y₁/Y₃ may form an epoxide; and, at least one of Y₁, Y₂, Y₃, Y₄, Y₅and Y₆ when present, is not H; X₁, X₂, X₃, and X₄ are independentlyselected from the group consisting of: H, R, OH, —OR, —CO₂H, —CO₂R′, F,Br, Cl, I, —CN, —SO₃H, —OSO₃H, NO₂, NH₂, —NHR, and —NR₂; where R is alinear, branched, or cyclic, saturated or unsaturated one to ten carbonalkyl group that is unsubstituted or is substituted with one or more of:OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR′, —NR′₂, NO₂, —CO₂H, —CO₂R′, andepoxide; and R′ is a linear, branched, or cyclic, saturated orunsaturated one to ten carbon alkyl group that is unsubstituted orsubstituted with one or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR″,—NR″₂, NO₂ and —CO₂H where R″ is a linear, branched, or cyclic,saturated or unsaturated one to ten carbon alkyl group; providing thatthe compound does not have the precise structure of pelorol or any oneof the group of structures consisting of:


2. The compound of claim 1, wherein Y₁-Y₆ are independently selectedfrom H, F, Br, Cl and I.
 3. The compound of claim 1, wherein Q is —CH₂—,—CH₂CH₂—, —CH═CH—, —CH₂—CH₂CH₂—, or —CH═CHCH₂—.
 4. The compound of claim1, wherein the carbon skeleton of Q is saturated.
 5. The compound ofclaim 1, wherein the carbon skeleton of Q consists of one or two carbonatoms.
 6. The compound of claim 1, wherein R₁ is methyl, ethyl, —CH₂OH,or —CH₂OR′.
 7. The compound of claim 1, wherein R₂ is methyl, ethyl,—CH₂OH, or —CH₂OR′.
 8. The compound of claim 1, wherein R′ in R₁ islimited to methyl, ethyl, propyl or butyl.
 9. The compound of claim 1,wherein R′ in R₂ is limited to methyl, ethyl, propyl or butyl.
 10. Thecompound of claim 8, wherein R′ is limited to methyl or ethyl.
 11. Thecompound of claim 1, wherein X₁ is H, OH, R, OR, —CONH₂, —CONHR′, or—COR′₂.
 12. The compound of claim 1, wherein X₂ is H, OH, R, OR, —CONH₂,—CONHR′, or —COR′₂.
 13. The compound of claim 1, wherein X₃ is H, OH, R,OR, —CONH₂, —CONHR′, or —COR′₂.
 14. The compound of claim 1, wherein Rand R′ in one or more of X₁, X₂, and X₃ are limited to methyl, ethyl,propyl and butyl.
 15. The compound of claim 1, wherein X₁ is H, OH, or—OCH₃.
 16. The compound of claim 1, wherein X₂ is H, OH, or OCH₃. 17.The compound of claim 1, wherein X₂ is H, OCH₃, or —NHOCH₃.
 18. Thecompound of claim 1, wherein X₃ is H, OH, or OCH₃.
 19. The compound ofclaim 1, wherein X₄ is H, R, OH, OR, CO₂H or CO₂R′.
 20. The compound ofclaim 1, wherein R and R′ in X₄ are limited to methyl, ethyl, propyl orbutyl.
 21. The compound of claim 1, wherein X₄ is H, R, OH, OCH₃, —CO₂Hor —CO₂CH₃.
 22. The compound of claim 1, selected from: homopelorol,dimethoxypelorol, PNSR-4A, PNSR-15A, PNSR-16A, PNSR-17A and PNSR-18A.23. The compound of claim 1, having the configuration S, R, R, S at C-5,C-8, C-9 and C-10 respectively.
 24. The compound of claim 1, having theconfiguration R, S, S, R at C-5, C-8, C-9 and C-10 respectively.
 25. Thecompound of claim 1, for use as a modulator of SHIP 1 activity.
 26. Thecompound of claim 25, wherein the compound is an agonist of SHIP 1activity.
 27. (Canceled)
 28. A method of making a compound of Formula IAin which R₁-R₄, X₁, X₃ and X₄ are as defined in claim 1,

wherein, L′ is a C₁-C₄ saturated or unsaturated alkyl linking group; andA is an activating group; comprising reacting a compound of Formula IIAor IIB:

in which L is absent or is a C₁-C₃ saturated or unsaturated alkyllinking group and E and E′ are electrophilic reactive groups; with acompound of Formula III

in which Nu is a group that renders the compound of Formula IIInucleophilic at Nu, followed by optional reduction and by hydrolysis, toproduce a compound of Formula IV

and condensing the compound of Formula IV to produce a compound ofFormula IA.
 29. The method of claim 28, wherein the compounds of FormulaIIA and IIB have the structures:


30. The method of claim 29, wherein the compounds of Formula IIA and IIBhave the structures


31. The method of claim 28, wherein the compound of Formula IIA or IIBis sclareolide or is derived from sclareolide.
 32. The method of claim28, wherein Nu is lithium.
 33. The method of claim 28, wherein A is OCH₃or —NHOCH₃.
 34. A pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more compounds of FormulaI or pharmaceutically acceptable salts thereof,

wherein; R₁ and R₂ are independently selected from the group consistingof: —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; R₃ and R₄are independently selected from the group consisting of: H, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; Q is a carbonskeleton selected from the group consisting of: —CH₂—, —CY₁Y₂—,—CH₂CH₂—, —CH═CH—, —CY₁Y₂CY₃Y₄—, —CH₂CH₂CH₂—, —CH═CHCH₂—, —CH═CHCY₁Y₂—,and —CY₁Y₂CY₃Y₄CY₅Y₆—; where Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆ areindependently selected from the group consisting of: H, F, Br, Cl, I,OH, OR′, and SH; or any one group of Y₁/Y₂, Y₃/Y₄, and Y₅/Y₆ may be ═O;or Y₁/Y₃ may form an epoxide; and, at least one of Y₁, Y₂, Y₃, Y₄, Y₅and Y₆ when present, is not H; X₁, X₂, X₃, and X₄ are independentlyselected from the group consisting of: H, R, OH, —OR, —CO₂H, —CO₂R′, F,Br, Cl, I, —CN, —SO₃H, —OSO₃H, NO₂, NH₂, —NHR, and —NR₂; where R is alinear, branched, or cyclic, saturated or unsaturated one to ten carbonalkyl group that is unsubstituted or is substituted with one or more of:OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR′, —NR′₂, NO₂, —CO₂H, —CO₂R′, andepoxide; and R′ is a linear, branched, or cyclic, saturated orunsaturated one to ten carbon alkyl group that is unsubstituted orsubstituted with one or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR″,—NR″₂, NO₂ and —CO₂H where R″ is a linear, branched, or cyclic,saturated or unsaturated one to ten carbon alkyl group.
 35. Thepharmaceutical composition of claim 34, wherein the one or morecompounds of Formula I is not solely pelorol.
 36. The pharmaceuticalcomposition of claim 34, comprising pelorol.
 37. The pharmaceuticalcomposition of claim 34, comprising a compound according to claim
 1. 38.A method of prophylaxis or treatment of an immune, hematopoietic,inflammatory or neoplastic disorder or condition comprisingadministering to a patient in need of said prophylaxsis or treatment, aneffective amount of a pharmaceutical composition according to claim 34.